PROJECT SUMMARY: Colorectal cancer (CRC) remains the 3rd most common cause of cancer-related death in the United States. Treatment depends on location and stage and often includes radiation, chemotherapy and surgery. CRC phenotype and response to therapy vary significantly. CRCs arise in part due to the accumulation of somatic mutations and Copy Number Variants (CNV's). No particular change has been found that fully predicts phenotype. We and others have identified genetic sub-clones in CRC. In other tumor types, recent studies have highlighted the potential role of genetic heterogeneity in tumor phenotype, but in CRC the role of heterogeneity and tumor sub-clones in tumor progression and response to therapy remains unknown. Thus, we hypothesize the following: Substantial intra-tumoral genetic heterogeneity is common in primary CRCs, and the distinct sub-clones present in a primary lesion underpin CRC biological behavior and clinical phenotype. Distinct sub-clones in the primary lesion give rise to CRC lymph node (LN) and distant metastases. The sub- clones present in a CRC lesion vary in their response to chemotherapy and radiation therapy. Three specific aims are proposed to address the overarching hypotheses: Specific Aim 1: We will characterize the extent and nature of genetic sub-clones in CRCs by studying somatic genetic alterations in multiple, distinct geographic regions from each tumor used in aims 2 and 3. We will use bioinformatics tools to integrate mutations and CNV's to define genetic sub-clones within each tumor and compare sub-clones across tumors. Specific Aim 2: To compare sub-clones in primary CRC's to those in patient-matched LN metastases in 20 patients with the goal of defining the role of sub-clonal populations in metastatic progression. The questions we propose to answer are these: 1. Are the sub-clones that metastasize to the LNs from the majority or minority clones in the primary tumor? 2. Are individual LN metastasis made up of single or multiple sub-clones? 3. When there are multiple LN metastasis in an individual patient, are they derived from the same sub-clone from the primary tumor or multiple different sub-clones? Specific Aim 3A: To assess relationships between the sub-clones in the tumor and the response of rectal cancer patients to pre-operative chemotherapy and radiation, by analyzing 40 matched primary rectal cancers before and after treatment. We hypothesize that some rectal cancer sub- clones are sensitive and others resistant to chemo- and radiation therapy. Aim 3B: In parallel, we will create xenograft models of 20 of the rectal cancers and assess whether they mimic the response to standard chemotherapy and radiation seen in the matched patients. This proposal will utilize innovative sequencing and bioinformatics techniques along with patient samples and clinical information. Ultimately, greater understanding of the genetic mechanisms underlying CRC progression and response to treatment will allow novel, targeted therapies to be proposed and tested. This project, the multidisciplinary mentorship team, and educational plan will prepare the candidate to be a fully independent investigator in the field of colorectal cancer genetics.